Bacterial pathogens cause devastating diseases, and it is imperative to improve our ability to treat bacterial infections. Among the most potent drugs in current use are the fluoroquinolone antibiotics, which trap the normally transient covalent intermediate formed by DNA gyrase with DNA, to create local DNA damage that is cytotoxic. In addition to gyrase, which is a type 2A topoisomerase, most bacteria also contain a structurally distinct type 1A topoisomerase, Topoisomerase 1 (Topo I). Like DNA gyrase, Topo I forms a transient covalent DNA adduct and should in principle be an excellent drug target, but no drugs currently target Topo I. Discovery and optimization of drugs that target Topo I and other topoisomerases has been limited by the lack of a mechanism-based assay for induction of topoisomerase-DNA adducts. We recently developed an assay, the RADAR assay (Rapid Assay of the DNA Adduct Response), that quantifies the covalent topoisomerase-DNA adducts formed as signature DNA damage in cells treated with topoisomerase poisons. We hypothesize that the RADAR assay will be useful for identifying new drugs that function by this mechanism, and for quantifying the potency of topoisomerase poisons in current use. The goal of this application is to validate the RADAR assay for screening compound libraries for drugs that poison bacterial topoisomerases. To achieve this, we will address current challenges in three aims. We will (1) show that DNA adducts formed by M. tuberculosis Topo I are toxic in mycobacteria and can be detected by the RADAR assay; (2) adapt and optimize the RADAR assay for high throughput screening; and (3) validate the RADAR assay for drug discovery by screening the TB Alliance TB Active Collection library. Impact: Validation of the RADAR assay will enable its application to discovery of new mechanism- based drugs to treat TB and a wide range of other infectious diseases.